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Amine-reactive fluorene probes: synthesis, optical characterization, bioconjugation, and two-photon fluorescence imaging.

Morales AR, Schafer-Hales KJ, Marcus AI, Belfield KD - Bioconjug. Chem. (2008)

Bottom Line: The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described.The conjugates displayed high chemical stability and photostability.The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The College of Optics and Photonics, University of Central Florida, P.O. Box 162366, Orlando, Florida 32816-2366, USA.

ABSTRACT
With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.

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Normalized UV−visible absorbance and fluorescence emission spectra of the amine-reactive probe 1 (—) and cyclic peptide conjugate 7 (---) in DMSO λmaxabs = 355 nm, λmaxem = 460 nm, Qy = 0.92.
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fig2: Normalized UV−visible absorbance and fluorescence emission spectra of the amine-reactive probe 1 (—) and cyclic peptide conjugate 7 (---) in DMSO λmaxabs = 355 nm, λmaxem = 460 nm, Qy = 0.92.

Mentions: The linear absorption and emission spectra of cyclic peptide-conjugate 7 in DMSO are shown in Figure 2. Conjugate 7 displayed absorption peak at 352 nm, while the emission maximum shifted from 400 nm for 1 to 460 nm for 7. In addition, the fluorescence quantum yield of 7 increased to 0.9 after conjugation.


Amine-reactive fluorene probes: synthesis, optical characterization, bioconjugation, and two-photon fluorescence imaging.

Morales AR, Schafer-Hales KJ, Marcus AI, Belfield KD - Bioconjug. Chem. (2008)

Normalized UV−visible absorbance and fluorescence emission spectra of the amine-reactive probe 1 (—) and cyclic peptide conjugate 7 (---) in DMSO λmaxabs = 355 nm, λmaxem = 460 nm, Qy = 0.92.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651597&req=5

fig2: Normalized UV−visible absorbance and fluorescence emission spectra of the amine-reactive probe 1 (—) and cyclic peptide conjugate 7 (---) in DMSO λmaxabs = 355 nm, λmaxem = 460 nm, Qy = 0.92.
Mentions: The linear absorption and emission spectra of cyclic peptide-conjugate 7 in DMSO are shown in Figure 2. Conjugate 7 displayed absorption peak at 352 nm, while the emission maximum shifted from 400 nm for 1 to 460 nm for 7. In addition, the fluorescence quantum yield of 7 increased to 0.9 after conjugation.

Bottom Line: The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described.The conjugates displayed high chemical stability and photostability.The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The College of Optics and Photonics, University of Central Florida, P.O. Box 162366, Orlando, Florida 32816-2366, USA.

ABSTRACT
With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.

Show MeSH
Related in: MedlinePlus